Project Summary The molecular basis of M. tuberculosis virulence is still incompletely understood. Within the host, M. tuberculosis must respond to, and resist, a plethora of stresses. Recent studies using genetically defined bacterial mutants have identified several signal transduction systems as critical to sense and respond to the host environment, including two component systems, transmembrane serine threonine kinases, and proteolytic systems. Rip1 is an intramembrane metalloprotease (S2P class) required for M. tuberculosis virulence in the mouse. Intramembrane proteases are membrane bound proteases with active sites in the membrane which cleave transmembrane substrates. Rip1 has four known substrates, the membrane spanning anti-sigma factors for SigK,L,M and D. However, the severe virulence defect of M. tuberculosis Drip1 is not phenocopied by an M. tuberculosis DsigKLM triple mutant or DsigD, raising the likelihood that additional downstream pathways controlled by Rip1 are the critical mediators of virulence. We have determined that Rip1 is required for resistance to several stresses relevant to pathogenesis, including copper, nitric oxide, and hypoxia. However, these phenotypes are independent of the four Rip1 controlled sigma factor pathways. Genetic epistasis analysis indicates that the copper and NO resistance pathway controlled by the Rip1 protease is independent of known copper efflux and Cu/NO induced transcriptional systems, indicating a novel mechanism of metal/NO resistance. Surprisingly, loss of anti-SigD (but not SigD, see above), a Rip1 substrate, causes severe copper sensitivity, suggesting an independent signaling role for this anti-Sigma, independent of its cognate sigma. We have isolated spontaneous mutants in the Drip1 background that suppress the Drip1 copper sensitivity phenotype and, by counterscreening these mutants against other stresses, whole genome sequencing, and complementation, have identified the PdtaS/PdtaR sensor kinase/response regulator pair as mediator of the Rip1 dependent copper and NO resistance. Transcriptional profiling reveals that the Rip1/PdtaS/PdtaR system controls, through transcriptional repression, stress induced expression of an operon encoding a nonribosomal peptide synthase recently shown to direct synthesis of a copper chelating peptide (a chalkophore), suggesting a plausible direct link to copper homeostasis. We also find that the virulence defect of the Drip1 strain is substantially reversed in NOS2 deficient mice, confirming that sensitivity to nitric oxide is the a contributor to the attenuation of the Drip1 strain. Based on these novel findings, we propose an experimental program to dissect the molecular mechanisms by which this newly discovered M. tuberculosis Rip1/PdtaS/PdtaR signaling cascade controls a stress response pathway that integrates cellular response to copper, nitric oxide, and hypoxia, the contribution of chalkophore biosynthesis to this pathway, and the role of this pathway in M. tuberculosis virulence. Characterization of this pathway through the following specific aims will yield significant insight into M. tuberculosis host-pathogen interactions.